EP3840751A1 - Nicht-peptid-opioid-rezeptormodulatoren - Google Patents

Nicht-peptid-opioid-rezeptormodulatoren

Info

Publication number
EP3840751A1
EP3840751A1 EP19851106.5A EP19851106A EP3840751A1 EP 3840751 A1 EP3840751 A1 EP 3840751A1 EP 19851106 A EP19851106 A EP 19851106A EP 3840751 A1 EP3840751 A1 EP 3840751A1
Authority
EP
European Patent Office
Prior art keywords
nfp
mor
compound
opioid
compounds
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19851106.5A
Other languages
English (en)
French (fr)
Other versions
EP3840751A4 (de
Inventor
Yan Zhang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Virginia Commonwealth University
Original Assignee
Virginia Commonwealth University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Virginia Commonwealth University filed Critical Virginia Commonwealth University
Publication of EP3840751A1 publication Critical patent/EP3840751A1/de
Publication of EP3840751A4 publication Critical patent/EP3840751A4/de
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D489/00—Heterocyclic compounds containing 4aH-8, 9 c- Iminoethano-phenanthro [4, 5-b, c, d] furan ring systems, e.g. derivatives of [4, 5-epoxy]-morphinan of the formula:
    • C07D489/06—Heterocyclic compounds containing 4aH-8, 9 c- Iminoethano-phenanthro [4, 5-b, c, d] furan ring systems, e.g. derivatives of [4, 5-epoxy]-morphinan of the formula: with a hetero atom directly attached in position 14
    • C07D489/08—Oxygen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D489/00—Heterocyclic compounds containing 4aH-8, 9 c- Iminoethano-phenanthro [4, 5-b, c, d] furan ring systems, e.g. derivatives of [4, 5-epoxy]-morphinan of the formula:
    • C07D489/06—Heterocyclic compounds containing 4aH-8, 9 c- Iminoethano-phenanthro [4, 5-b, c, d] furan ring systems, e.g. derivatives of [4, 5-epoxy]-morphinan of the formula: with a hetero atom directly attached in position 14

Definitions

  • This invention generally relates to non-peptide compounds that modulate opioid receptor activity.
  • the compounds are modulators of the MOR opioid receptor with predominantly central activity, and are used to treat e.g. opioid addiction.
  • Opioid receptors belong to the class A Rhodopsin-like G-protein coupled receptor
  • GPCR family and can be classified into four subtypes known as the m opioid receptor (MOR), K opioid receptor (KOR), d opioid receptor (DOR), and nociception opioid peptide (NOP) receptor.
  • MOR is the main pharmacological target for opioid medications, such as morphine. Interaction between the MOR and G(Xj /0 leads to activation of the MOR which results in opening of inwardly rectifying K + (GIRK) channels, inhibition of the voltage-gated Ca 2+
  • VGCC vascular endothelial growth factor
  • MOR activation include beneficial antinociception and MOR agonists have been widely used for treating moderate to severe pain.
  • opioid-induced constipation OIC
  • respiratory depression OIC
  • addiction opioid-induced constipation
  • opioid addiction/abuse has become a global epidemic. It is reported that over 33,000 people died due to opioid overdose in the US alone in 2015, a more than 14 fold increase over the previous 20 years.
  • opioid abuse increases the prevalence of diseases such as HIV, tuberculosis, and hepatitis, especially for users who inject these drugs.
  • naltrexone used for long term treatment
  • naloxone used for short-acting treatment. While these opioid antagonists do not exhibit the side effects of opioid agonists, such as addiction and respiratory suppression, high doses of naltrexone and naloxone may induce hepatotoxicity and
  • cardiovascular and pulmonary problems cardiovascular and pulmonary problems.
  • interactions between these compounds and the DOR or KOR may induce mood changes or block transmission of neurotransmitters.
  • NFP produced significantly fewer withdrawal symptoms than naloxone at similar doses.
  • these compounds can be used to treat opioid abuse and addiction.
  • R , R 2 , R 3 and R4 are independently selected from the group consisting of hydrogen, halogen, a substituted or unsubstituted C 1 -C 5 alkyl (e.g., a halogenated C 1 -C 5 alkyl), C 1 -C 5 alkoxy, a substituted or unsubstituted aryl or heteroaryl, a carboxyl, an alkoxycarbonyl, an acyl, sulfonic acid, a sulfonyl, an aminocarbonyl, CN, or N0 2 .
  • n can be 0, 1, 2, or 3.
  • the compound of formula I is used to modulate opioid receptors.
  • the compound of formula I is used to selectively modulate the MOR opioid receptor.
  • the compound of formula I acts as a modulator to MOR and can be used for the treatment of opioid abuse and addiction, alcoholism and neurological disorders associated with opioid receptors.
  • the compound of formula I is NFP
  • the compound of formula I is NYP:
  • FIG. 1 A and B. Dose-response studies of NFP (A) and NYP (B) as antagonists in mice.
  • FIG. 5A and B MOR partial agonism and antagonism by NFP in mMOR-CHO cells.
  • Ligand concentration-effect for stimulation of [ 33 S]GTPyS binding in membranes from mMOR-CFIO cells were determined alone (A) or in the presence of 0.3 mM DAMGO (B).
  • FIG. 6 A and B.
  • A The chemical structure of NFP; B, atom notations from the docking studies.
  • FIG. 7A and B Calcium mobilization assay of NFP as an agonist (A) or an antagonist (B).
  • the EC50 of DAMGO is 36.32 ⁇ 1.85 nM
  • IC 50 values of naltrexone and NFP are 6.62 ⁇ 1.45 nM and 76.09 ⁇ 2.50 nM, respectively.
  • Figure 8 The result of bidirectional transport assay for NFP.
  • P app values of tested compounds in apical-to-basolatoral (A-B) and basolateral-to-apical (B-A) directions were determined by incubating Caco-2 monolayers at 37 °C for 2 h with NFP (20 mM). Data represent means ⁇ SD.
  • Figure 9A and B The result of the warm-water tail-withdrawal test.
  • NAP peripherally acting NAP and N-((4R,4aS,7R,7aR,l2bS)-3-(cyclopropylmethyl)-4a,9- dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-lH-4,l2-methanobenzofuro[3,2-e]isoquinolin-7-yl)-3- methylisonicotinamide (NMP); (see Figure 10), these compounds were found to be centrally acting. In addition, they caused fewer withdrawal effects than naloxone.
  • the compounds that were synthesized may find application in the treatment of opioid addiction, as well as in the treatment of other neurological disorders implicating opioid receptors (e.g. alcoholism, some psychiatric disorders, etc.).
  • a“modulator” is a chemical compound that acts on a receptor e.g. as an antagonist or agonist or a partial agonist, etc.
  • an antagonist that binds the receptor may interfere with and/or inhibit its physiological activity.
  • a partial agonist binds to and activates a receptor but has a lower efficacy than a full agonist.
  • the compounds provided herein, which include pharmaceutically acceptable salts and solvates, have a general formula as shown in Formula 1:
  • Ri, R 2 , R 3 and R4 are independently selected from the group consisting of hydrogen, halogen, a substituted or unsubstituted C1-C5 alkyl (e.g., a halogenated C1-C5 alkyl), C1-C5 alkoxy, a substituted or unsubstituted aryl, a carboxyl, an alkoxycarbonyl, an acyl, sulfonic acid, a sulfonyl, an aminocarbonyl, CN, or N0 2 .
  • n can be 0, 1, 2, or 3.
  • a compound of the formula I, pharmaceutically acceptable salt, or solvate thereof is provided, where, n is 0 and Ri, R 2 and R4 are hydrogen.
  • a compound of the formula I, pharmaceutically acceptable salt, or solvate thereof is of the formula:
  • R 2 is F, CN, NO?, CF 3 , COOH, COOCl3 ⁇ 4, C 2 H 5 -, isobutyl, cyclopentyl, cyclohexyl, cycloheptyl or phenyl.
  • the compound of formula I has any of these exemplary formulas:
  • the compound of formula I is used to modulate opioid receptors.
  • the compound of formula I is used to selectively interact with MOR.
  • the compound of formula I acts as a modulator and is useful for the treatment of opioid abuse and addiction, alcoholism, and/or neurological disorders associated with opioid receptors.
  • a“compound” of the invention includes all solvates, complexes, polymorphs, radiolabeled derivatives, tautomers, stereoisomers and optical isomers of the compounds of Formula I and salts thereof.
  • the compounds described herein are generally delivered (administered) in a
  • compositions generally comprise at least one of the disclosed compounds, i.e. one or more than one (a plurality of) different compounds (e.g. 2 or more) in a single formulation.
  • the compositions also generally include a pharmacologically suitable (physiologically compatible) carrier, which may be aqueous or oil-based.
  • pharmacologically suitable (physiologically compatible) carrier which may be aqueous or oil-based.
  • such compositions are prepared as liquid solutions or suspensions, or as solid forms such as tablets, pills, powders and the like.
  • Solid forms suitable for solution in, or suspension in, liquids prior to administration are also contemplated (e.g. lyophilized forms of the compounds), as are emulsified preparations.
  • the liquid formulations are aqueous or oil-based suspensions or solutions.
  • the compounds are mixed with excipients which are pharmaceutically acceptable and compatible with the compounds, e.g. pharmaceutically acceptable salts.
  • Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof.
  • the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, preservatives, and the like. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like are added.
  • the composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration.
  • the final amount of compound in the formulations varies, but is generally from about 1-99%. Still other suitable formulations for use in the present invention are found, for example in Remington's Pharmaceutical Sciences, 22nd ed. (2012; eds. Allen, Adejarem Desselle and Felton).
  • materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as TWEEN® 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose
  • glycols such a propylene glycol or polyethylene glycol
  • esters such as ethyl oleate and ethyl laurate
  • agar buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
  • “Pharmaceutically acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention.
  • acid addition salts can be prepared in situ during the final isolation and purification of the compounds.
  • acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.
  • Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis- .beta.-hydroxynaphthoates, gentisates, isethionates, di-p-tol
  • Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed.
  • Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred.
  • Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like.
  • Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia,
  • ethylenediamine N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'- dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium
  • hydroxide triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and
  • compositions may be administered in vivo by any suitable route including but not limited to: inoculation or injection (e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural, intraarticular, intramammary, and the like), topical application (e.g. on areas such as eyes, skin, in ears) and by absorption through epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal, rectal, gastrointestinal mucosa, and the like).
  • suitable means include but are not limited to: inhalation (e.g. as a mist or spray), orally (e.g.
  • the mode of administration is oral or by injection.
  • An individual who is treated using the compounds described herein generally is a subject who is or has previously been addicted to opioids, either as the result of a legitimate medical use (e.g. the treatment of pain using a prescription opioid such as OXYCONTIN®, VICODIN®, etc.) and/or through recreational use.
  • the person may be addicted to opioids, or may be at risk of becoming addicted due to, e.g., long-term usage, or may have previously been addicted and is in danger of a relapse.
  • predisposition and personality factors, including ADD, OCD, bipolar disorder, schizophrenia and depression; may all lead to increased susceptibility to addiction, which can be addressed or averted by intervention using the compounds described herein.
  • the amount of a compound that is administered to an individual will vary based on several factors, as will be understood by those of skill in the art.
  • the dose and frequency of administration may vary according to the gender, age, weight, general physical condition, ethnic background, etc. of the individual, as well as whether or not the individual has other diseases or conditions that might impinge on the treatment.
  • the dose for a therapeutically effective amount will be in the range of from about 0.01 to about 1000 mg/kg of body weight.
  • a therapeutically effective amount is generally an amount that ameliorates, lessens or improves at least one symptom of the disease/condition that is being treated, and this amount may also eradicate all symptoms of the disease/condition, i.e., it may cure the subject of the disease/condition. In particular, the subject may become entirely free of the craving for opioids and addiction or relapse may be prevented.
  • the compositions may be administered in conjunction with other treatment modalities such as substances that treat pain or addiction, for example: with opioids e.g.
  • Compounds 1-6 were derivatives with electron-withdrawing groups at the 3’ position on the pyridyl ring with a corresponding increment in size of the substituents.
  • Compounds 7-12 were derivatives with electron-donating groups at the 3’ position on the pyridyl ring with different bulkiness.
  • the 3’ -substituted pyridyl carboxylic acids were from commercial sources.
  • the 3’-substituted pyridyl carboxylic acids were prepared as shown in Scheme 1 (Yakhontov, et al. Chem. Heterocyclic Com. 1967, 3, 829-831; Xin, J Chem. Res. 2008, 2008, 412-415; Okawa, et al. J Med. Chem. 2017, 60, 6942-6990).
  • a competition radioligand binding assay was used to determine the binding affinity and selectivity of the new NAP derivatives for the MOR, KOR, and DOR.
  • Chinese hamster ovary (CHO) cell lines expressing monocloned opioid receptors were used for this assay.
  • the MOR was labeled with [ 3 H]naloxone, while KOR and DOR were both labeled with
  • the isobutyl group on the pyridyl ring of compound 8 may result in steric clashes or unfavorable interactions with hydrophilic residues in the binding pocket that would decrease the binding affinity of compound 8 to the MOR.
  • compounds 9-12 carried even bigger substituents on the pyridyl ring while maintained high binding affinities to the MOR at the subnanomolar level.
  • One possible explanation of such an observation could be that some significant conformation changes induced by the much larger substituents on the pyridyl ring (compounds 9-11) may faciliate the compounds to interact with the binding pocket in the MOR in a more favorable fashion.
  • the 3:, S-GTP[YS] functional assay was then conducted to determine the potency and relative efficacy of the synthesized compounds at the MOR.
  • the potency was determined as the EC 50 while the efficacy was determined as the E ma x relative to DAMGO, a MOR full agonist. For the results, it seemed that all the compounds acted as partial agonists with moderate efficacy.
  • NAP derivatives were further assessed for their acute agonistic and/or antagonistic effects using the tail flick assay in mice as previously reported. 44, 37 Briefly, the derivatives were studied for their ability to produce antinociception or block the antinociception produced by morphine (10 mg/kg). As shown in Figure 1 A, none of these new derivatives showed significant antinociception compared to morphine at the same dose (10 mg/kg). However, when they were tested as antagonists at the same dose (Figure IB, 10 mg/kg), compounds 1 (NFP) and 2 (NYP) antagonized morphine’s antinociception effect significantly.
  • the percent maximum possible effects (%MPEs) of morphine (10 mg/kg) in the presence of NFP and NYP (10 mg/kg) were only 6.2 ⁇ 2.4% and 7.8 ⁇ 5.4%, respectively.
  • the antagonism of NFP and NYP were shown to be dose-dependent ( Figure 2A and 2B).
  • other NAP derivatives did not significantly block morphine’s antinociception at the tested doses of 10 mg/kg.
  • NFP and NYP crossed the BBB and antagonized morphine’s antinociception, while NAP was identified as a P-glycoprotein substrate and failed to cross the BBB effectively.
  • fluoro and nitrile groups on the pyridyl ring may play an important role in improving their CNS penetration. It is understood that about 5-15 % of drugs on the market are fluorinated compounds 39, 60 and it has been shown that these compounds have improved metabolic stability and physicochemical properties. Meanwhile fluorination seems to enhance the CNS penetration ability of non-CNS drugs and the efflux function of P-glycoprotein. 61, 53 This may help explain why NFP crossed the BBB while NAP did not.
  • nitrile groups may also enhance metabolic stability and improve hydrogen bonding interactions with residues in the orthosteric binding site of protein targets. “ Currently, there are at least 30 drugs containing nitrile groups on the market and more than 20 drug candidates containing nitrile groups under clinical trials. 62 An example of such a drug is piritramide, an opioid analgesic which crosses the BBB and is used for post-operation pain like morphine. 63
  • Opioid antagonists such as naloxone and naltrexone have been applied to reverse the effects of opioid agonists in cases of opioid overdose and in opioid addiction treatments.
  • NFP and NYP were identified as opioid antagonists in vivo, a withdrawal study was conducted using morphine-pelleted mice to determine whether these two compounds produced withdrawal effects similar to naloxone. In this study, somatic symptoms of opioid withdrawal (shakes, jumps, and paw tremors) were quantified over a period of 20 minutes. 33 As shown in Figure 3, NFP produced significantly fewer wet dog shakes, jumps, and paw tremors at 10 mg/kg than naloxone at 1 mg/kg. Meanwhile, at much higher doses (50 and
  • NFP produced wet dog shakes (Figure 3A) and paw tremors (Figure 3 C), but not significantly different from those of naloxone at 1 mg/kg. More interestingly, NFP at a high dose of 50 mg/kg still produced significantly fewer jumps ( Figure 3B) than naloxone at 1 mg/kg.
  • NYP produced significantly fewer wet dog shakes and jumps at 1 mg/kg than naloxone at 1 mg/kg, but at doses 5 mg/kg and higher it produced wet dog shakes (Figure 4A) and jumps (Figure 4B) similar to naloxone at 1 mg/kg. Also, NYP produced similar paw tremors (Figure 4B) at 1 mg/kg similar to naloxone at 1 mg/kg.
  • NFP has fewer significant withdrawal effects compared to naloxone.
  • the in vitro and in vivo results obtained for NFP suggest that NFP is a MOR/KOR ligand with the potential to be applied in opioid addiction treatments.
  • NFP partial agonism and antagonism via concentration- effect curves for stimulation of [ 3:, S]GTPYS binding in mMOR-CHO cells by NFP and DAMGO alone (Figure 5A) in comparison to NFP curves in the presence in an EC90 DAMGO
  • NFP and naltrexone inhibited DAMGO-stimulated [ 35 S]GTPYS binding with Kj values of 0.86 ⁇ 0.09 and 0.67 ⁇ 0.05 nM, similar to their K j values for MOR binding (Table 1). NFP had a relative efficacy that was approximately 35% of DAMGO and 3- fold that of naltrexone under these experimental conditions.
  • the antagonist-bound crystal structures of MOR (PDB ID: 4DKL) 64 , KOR (PDB ID: 4DJH) 65 and DOR (PDB ID: 4EJ4) 66 were downloaded from Protein Data Bank http://www.rcsb.org and NFP was docked into the three receptors by GOLD 5.4 (not shown).
  • the binding poses of NFP with the highest CHEM- PLP scores from the docking studies were chosen as the optimal binding poses of the ligand in the MOR, KOR, and DOR.
  • NFP was designed and synthesized according to the ‘message-address’ concept where the‘message’ moiety (epoxymorphinan moiety) was assumed to determine its efficacy and the‘address’ moiety (pyridyl ring) to contribute to its selectivity (Figure 6A). Comparing the binding poses of NFP in the MOR, KOR, and DOR with those of NAP, we found that the identical‘message’ moiety of NFP and NAP bound with the same domain of the MOR, KOR, and DOR through similar interactions with the conserved residues in the three receptors.
  • the epoxymorphinan moiety formed hydrophobic interactions with the conserved residues M 3 36 , W 6 48 and H 6 32 (superscript numbers follow the Ballesteros- Weinstein numbering method for GPCRs 69 ) and hydrogen bonding interactions with Y 3 33
  • the fluorine atom on the pyridyl ring (‘address’ moiety) of NFP is a strong electron withdrawing group which would weaken the capability of the nitrogen atom on the pyridyl ring of NFP to keep a proton compared to the case of NAP and NMP. 38
  • the nitrogen atom on the pyridyl ring of NFP might form electrostatic interactions with the conserved residue E229 3 33 in the MOR, D223 3 33 in the KOR, and D210 3 33 in the DOR.
  • the pyridyl ring of NFP could also form hydrophobic interactions with the conserved hydrophobic residue L ECL2 and F ECL2 (Table 3).
  • the‘address’ moiety (pyridyl ring) of NFP seemed to bind to the same domain (termed‘address’ domain) in the MOR, KOR, and DOR.
  • this‘address’ domain T2l 8 ECL2 , T225 3 3 I , and L232 3 38 in the MOR, S211 ECL2 , Y219 5 31 , and M226 5 38 in the KOR and Ml99 ECL2 , S206 5 31 , and T213 5 38 in the DOR, Figure 6
  • T2l 8 ECL2 , T225 3 3 I , and L232 3 38 in the MOR S211 ECL2 , Y219 5 31 , and M226 5 38 in the KOR and Ml99 ECL2 , S206 5 31 , and T213 5 38 in the DOR, Figure 6
  • the electrostatic interaction between the nitrogen atom on the pyridyl ring of NFP and E229 3 33 in the MOR was much stronger than those in the NFP/KOR and NFP/DOR complexes, which may help to explain the highest affinity of NFP to the MOR.
  • the non-conserved Y219 3 31 and S211 ECL2 could also form hydrogen bond with the nitrogen atom on the pyridyl ring of NFP, which may facilitate the binding of NFP to the KOR.
  • the third generation 6b-N-4 '-pyridyl substituted naltrexamine derivatives were designed, synthesized and evaluated in both in vitro and in vivo assays.
  • the in vitro competition assays showed that the third generation compounds carrying varies substitutions at the 3’ position of pyridyl ring carried a different selectivity profile than NAP.
  • the third generation compounds were typically MOR KOR dual-selective while NAP was highly MOR selective. Most of the new compounds retained their binding affinity to the MOR with subnanomolar level and one-digit nanomolar binding affinity to KOR. All of the compounds had low to medium efficacy at the MOR with one-digit nanomolar potencies.
  • NFP and NYP significantly antagonized the antinociception produced by morphine in a dose dependent fashion.
  • the in vivo withdrawal studies showed that NFP produced significantly less withdrawal symptoms than naloxone at similar doses.
  • hydrobenzotriazole (2.5 eq), 4 A molecular sieves, and trimethylamine (5 eq) were added to a solution of the carboxylic acid (2.5 eq) in DMF on an ice-water bath under N 2 protection. After 30 min, a solution of 6p-naltrexamine (1 eq) in DMF was added dropwise. The mixture was kept stirring overnight at room temperature and filtered the next day. The filtrate was then concentrated under reduced pressure to remove the solvent. The residue was dissolved in MeOH and potassium carbonate (2 eq) was added to the mixture. The reaction mixture was stirred overnight at room temperature. Next day, the mixture was concentrated and the residue was purified with silica gel column to obtain the target compound.
  • IR (diamond, cm 1 ) F ma 3111.05, 2087.54, 1721.23, 1626.30, 1501.34, 1467.64, 1371.61, 1322.77, 1079.34, 1031.03, 858.34, 731.40.
  • IR diamond, cnT 1
  • IR diamond, cnT 1
  • F max 3269.93, 2925.57, 2850.60, 1644.45, 1537.65, 1504.66, 1450.26, 1411.24, 1374.41, 1324.67, 1240.14, 1188.89, 1149.13, 1130.35, 1095.73, 1035.49, 984.59, 918.19, 881.92, 856.04, 828.64, 798.39, 785.52, 762.60, 745.35, 728.43, 702.73, 677.85.
  • Morphine morphine sulfate pentahydrate salt
  • naloxone a compound obtained by the National Institute of Drug Abuse (NIDA), Bethesda, MD and then made into a 10 mM stock solution by dissolving in distilled water which was further diluted to the desired concentrations. All other reagents were purchased from either Sigma-Aldrich or Thermo Fisher. Animals. Male Swiss Webster mice (25-30 g, Harlan Laboratories, Indianapolis, IN) were raised in animal care quarters and maintained at room temperature on light-dark cycle. Food and water were available ad libitum. Protocols and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Commonwealth University and complied with the recommendations of the IASP (International Association for the Study of Pain).
  • IACUC Institutional Animal Care and Use Committee
  • mice Swiss Webster mice were used for this experiment. Water bath temperature was maintained at 56 ⁇ 0.1 °C. The baseline latency (control) was determined before the test compound was injected subcutaneously (s.c.) into the mice. The average baseline latency obtained for this experiment was 3.0 ⁇ 0.1 s and only mice with a baseline latency of 2 to 4 s were used.
  • s.c. subcutaneously
  • the average baseline latency obtained for this experiment was 3.0 ⁇ 0.1 s and only mice with a baseline latency of 2 to 4 s were used.
  • test compound was given 5 min before morphine.
  • Tail immersion was conducted 20 min after giving morphine.
  • %MPE was calculated for each mouse using at least five mice per drug.
  • AD 50 values were calculated using the least-squares linear regression analysis followed by calculation of 95% confidence interval by Bliss method.
  • GOLD 5.4 with default settings was used to conduct the docking study.
  • Automated docking was conducted with a distance constraint of 4 A between the nitrogen atom at the 1 T position of the epoxymorphinan nucleus and the conserved D .
  • a hydrogen bond constraint was applied between NFP’s dihydrofuran oxygen and the phenolic oxygen of the conserved Y 3 33 .
  • the highest scored solutions (CHEM-PLP) were selected and merged into the receptor to obtain the optimal binding poses of NFP in the MOR, KOR, and DOR.
  • the binding poses were energy minimized in Sybyl-X 2.0.7 with 5000 iterations under TAFF to remove the clashes and strain energy between NFP and the three receptors.
  • GPCRs G-protein coupled receptors
  • MOR m opioid receptor
  • KOR k opioid receptor
  • DOR 6 opioid receptor
  • FDA Food and Drug Administration
  • mMOR-CHO Chinese hamster ovary cell lines expressing mouse monocloned m opioid receptor (mMOR-CHO), Hank's balanced salt solution (HBSS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Phosphate buffered saline (PBS), paraformaldehyde (PFA), transepithelial electrical resistance (TEER), P- glycoprotein (P-gp), permeability directional ratio (PDR), basolateral-to-apical permeability (P app , B-A), apical-to-basolateral permeability (P app , A-B), percent maximum possible effect (%MPE), Chinese hamster ovary (CHO).
  • HBSS Hank's balanced salt solution
  • DMEM Dulbec
  • test compounds were then incubated with 50 pL/well loading buffer (6 mL assay buffer, 24 pL Fluo4-AM solution (Invitrogen), 12 pL probenecid solution) for 45 min. Following incubation, different concentrations of the test compounds were added by
  • FLEXSTATION ® 3 microplate reader (Molecular Devices) and read at ex494/em516. Each concentration was run in triplicate.
  • the cells were incubated with the same loading buffer as the agonist assay for 45 min. Then, different concentrations of the test compounds (20 pL/well) were manually added to each well followed by another 15 min incubation. After that, the solution of DAMGO in assay buffer (500 nM) or just assay buffer (blank) was added by
  • mMOR-CElO cells were grown in culture media (DMEM/F12 media, 10% FBS, 1% penicillin/streptomycin, 0.5% G418) for 5 days in an incubator set at 37 °C with 5% C0 2 and 95% humidity. On the fifth day when the cells were confluent, the culture media was removed and the cells were rinsed with 5 mL PBS. The cells were then treated with DAMGO (5 pM), morphine (5 pM), nalbuphine (1 pM), NFP (1 pM), naltrexone (1 pM) and vehicle (0.02% DMSO) dissolved in DMEM/F12 media and incubated for 24 h.
  • DAMGO 5 pM
  • morphine pM
  • nalbuphine (1 pM
  • NFP 1 pM
  • naltrexone naltrexone
  • phosphate-buffered saline PBS
  • 5 mL PBS was added to each dish and the cells were then scraped off the dishes using a scraper.
  • the cells were then centrifuged at 1,000 x g for 10 min. After centrifugation, the supernatant was decanted and membrane buffer (50 mM Tris, 3 mM MgCl 2 , and 1 mM EGTA, pH 7.4) was added to each sample.
  • the cells were then homogenized and centrifuged again at 50,000 x g for 10 min. The supernatant was decanted and the cells were homogenized again in membrane buffer.
  • a Bradford assay was conducted to determine the concentration of the membrane protein.
  • the membrane protein preparations were then stored at -80 °C.
  • Nonspecific binding was determined using 5 mM naltrexone. The incubation was terminated by rapid filtration and bound radioactivity was determined as described previously (Selley et ah, 1998). K O and B max values were determined by non-linear regression using GraphPad Prism 6.0
  • Ligand-stimulated [ 33 S]GTPyS binding was performed as described previously (Selley et al., 1998). Briefly, membranes from the treated mMOR-CHO cells (10 pg of protein) were incubated with 0.1 nM [ 35 S]GTPyS (specific radioactivity was 1250 Ci/mmol) and 20 pM GDP for 90 min at 30 °C with varying concentrations of DAMGO in assay buffer (50 mM Tris-HCl,
  • [ SJGTPyS binding was defined as ligand-stimulated minus basal binding.
  • N2A cells stably transfected with the rat MOR cDNA epitope-tagged with HA at N-terminus (N2A-HA-rMOR) were established previously (Obeng, et ah, 2019) and clones H38 and H16 expressing MOR at 1-2 pmole/mg protein were used in the study.
  • Cells were cultured in 10-cm dishes at 37°C with 5% C0 2 in humidified air in MEM (Minimum Essential Medium, ref 41500, Gibco, NY) supplemented with 10% FBS and penicillin, streptomycin and amphotericin (A5955, Sigma, MO) and grew to 80% confluence.
  • MEM Minimum Essential Medium, ref 41500, Gibco, NY
  • N2A-HA-rMOR cells were sub-cultured onto coverslips placed in 6-well plates at 300,000 cells per well. Forty-eight hours later, mouse anti-HA. l 1 antibodies (Clone 16B12, BioLegend, CA) were added at 1 : 1000 to cell medium and incubated for 1 h. NFP (final 10 mM) or vehicle was added and incubated for 15 min followed by addition of etorphine (final 10 mM) or vehicle and incubated for another 15 min.
  • Caco-2 cells (passage 48; ATCC, Manassas, VA) were plated on 12 mm, 0.4 mih, #3460 - Clear TRANS WELL ® inserts (Corning Incorporated, Corning, NY) at an initial seeding density of 90,000 cells/well.
  • the cells were cultured in DMEM supplemented with 10 % FBS and non-essential amino acids for 23 days. On day 24, the medium was removed and the transwells were rinsed with PBS.
  • HBSS buffer pH 7.4 was added to both apical (0.5 mL) and basolateral (1.5 mL) chambers and the initial transepithelial electrical resistance (TEER) values were recorded.
  • HBSS buffer in apical or basolateral chambers was replaced by NFP solutions in HBSS (20 mM) with or without P-glycoprotein (P-gp) inhibitor (elacridar, 1 mM) (Matsson et al., 2009). 200 pL aliquots were collected from the receiver chamber up to 2 h. Acetonitrile (50 pL) was added to each sample (50 pL), which was vortexed then centrifuged at 10,000g for 5 min at 20 ° C. The supernatant was then used for analysis by LC-MS as described below. At the end of 2 h, the solution in both chambers was replaced with HBSS and postexperiment TEER values were measured.
  • P-gp P-glycoprotein
  • the integrity of the monolayers was confirmed by studying the transport of lucifer yellow, a low permeability marker (Maharao et al. 2017)
  • the permeability directional ratio (PDR) was calculated as the ratio of basolateral-to-apical permeability to apical-to-basolateral permeability (P app, B-A / P app, A-B) ⁇
  • HYPERSILTM BDS Cl 8 column (50 x 4.6 mm, 3 pm; Waltham, MA) with isocratic elution (aq. 0.1% formic acid and acetonitrile, 85: 15). The flow rate was 1.0 niL/min and the temperature of the column was maintained at 35 °C. The injection volume was 40 pL.
  • the MS system consisted of an ACQUITY® QDA® mass spectrometer (Waters Corporation, Milford, MA) equipped with electrospray ionization in the positive ion detection mode. The run time for each sample was 5.20 min. Data acquisition and processing were performed using Empower 3 software (Waters Corporation, Milford, Massachusetts, USA). The retention time for NFP was found to be 2.99 min and had an m/z value of 466.20 ([NFP + H] + ). 2.5 Warm-Water Tail-Withdrawal Test
  • mice Male male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were individually housed in a temperature-controlled (20-22°C) AAALAC-accredited facility in which they had ad libitum access to food and water. The mice were maintained on a 12 h/12 h light-dark cycle (0600-1800 lights on) for the duration of the experiment and were tested during the light segment of this cycle. All subjects were acclimated to the vivarium for at least one week before the commencement of studies.
  • Apparatus and drugs A commercial warm water bath (Model # JBN5 US; Grant Instruments Ltd., Cambridge, UK) maintained at 52.5 ⁇ 0.5°C was used to assess nociception. Tail withdrawal latencies were measured with a digital stopwatch (Model # 14-649-7; Fisher Scientific, Pittsburgh, PA). Morphine sulfate was obtained from the NIDA Drug Supply Program. All compounds were dissolved in sterile saline (Fisher Scientific, Pittsburgh, PA; Cat. # 125EZ-7002) and were administered s.c. All compounds were administered in a volume equivalent to 10 mL/kg body weight. Technicians were blinded to treatment groups/
  • Day 1 Tests At the start of testing of Day 1, each mouse was placed in a restraint cloth fashioned from a surgical drape, and the distal 3 cm of its tail was submerged in the warm water bath to determine its baseline withdrawal latency. A digital stopwatch was used to record the amount of time that elapsed between tail immersion and tail withdrawal (i.e., tail-withdrawal latency). Immediately after that, mice received consecutive injections of saline (i.e., morphine's vehicle) and their scheduled NFP condition (i.e., vehicle or 0.3 mg/kg NFP), and were returned to their home cage.
  • saline i.e., morphine's vehicle
  • NFP condition i.e., vehicle or 0.3 mg/kg NFP
  • tail-withdrawal latencies were re-determined, and mice were immediately injected with the lowest dose of morphine (1 mg/kg). Following the 30-min pretreatment period, tail-withdrawal latencies were re-determined, and mice were immediately injected with the lowest dose of morphine (1 mg/kg). Following the 30-min pretreatment period, tail-withdrawal latencies were re-determined, and mice were immediately injected with the lowest dose of morphine (1 mg/kg). Following the 30-min pretreatment period, tail-withdrawal latencies were re-determined, and mice were immediately injected with the lowest dose of morphine (1 mg/kg). Following the 30-min pretreatment period, tail-withdrawal latencies were re-determined, and mice were immediately injected with the lowest dose of morphine (1 mg/kg). Following the 30-min pretreatment period, tail-withdrawal latencies were re-determined, and mice were immediately injected with the lowest dose of morphine (1 mg/kg). Following the 30-min pretreatment period, tail-withdrawal latencies
  • Days 2-6 On non-test days (i.e., Days 2-6) at a similar time each afternoon (between
  • mice received two injections one right after the other, either 10 mg/kg morphine + NFP vehicle (Vehicle Group) or 10 mg/kg morphine + 0.3 mg/kg NFP (NFP Group).
  • mice were re-assessed in the warm-water tail-withdrawal procedure with cumulative morphine dosese identical to that described for Day 1.
  • SJ-GTPyS binding assays directly measure the activation of G-proteins induced by a ligand binding to its receptor, whereas calcium mobilization assays measure the release of calcium as a readout of more downstream intracellular signaling cascade (Selly et al., 1998). After stimulated by a ligand, the conformation of the GPCR changes, thus activating its corresponding intracellular downstream messenger pathway. As a result of these variations, cytosolic calcium is released which can be measured using a fluorescent calcium indicator (Zhu et ah, 2008). Chinese hamster ovary (CHO) cell lines expressing the m opioid receptor were used for this assay. DAMGO and naltrexone were used as control compounds.
  • DAMGO opioid agonist
  • morphine opioid agonist
  • nalbuphine opioid partial agonist
  • naltrexone opioid antagonist
  • the DAMGO EC50 values of the cells treated with morphine and DAMGO increased about 6- and 4-fold, respectively, this indicates that morphine and DAMGO desensitized the MOR.
  • the DAMGO EC50 values of nalbuphine, naltrexone and NFP treated cells were similar to the DAMGO EC50 value for the vehicle treated cells (Table 5).
  • Naltrexone 3 3.86 ⁇ 0.68 3.84 ⁇ 0.44* 21 .85 ⁇ 0.78 126.73 ⁇ 3.97 5.80
  • NFP did not show significant desensitization and downregulation of the MOR.
  • NFP has the potential to be used therapeutically for opioid use disorder treatments without likely producing tolerance.
  • Desensitization and downregulation of opioid receptors may occur following prolonged binding of opioid agonists to the receptors, e.g. due to internalization via different mechanisms. Following internalization, receptors may be recycled or downregulated. NFP did not cause significant desensitization and downregulation of the MOR. We examined if NFP promoted MOR internalization. Two different N2A-HA-rMOR-N2A cell lines were used for this assay because CFIO cells have a small cytosolic volume and it is difficult to visualize receptor internalization. The opioid agonist etorphine was used as a positive control.
  • NFP showed evident MOR antagonism in in vivo studies with an AD50 value of 2.82 (1.34-5.94) mg/kg with a 95% confidence level (CL). Additionally, NFP precipitated dramatically fewer withdrawal symptoms compared with naloxone at the same doses. These characteristics were maintained even at the highest dose (100 mg/kg) tested.
  • NFP was shown to act as a CNS partial agonist (Example 1) without apparent desensitization and downregulation effects. Moreover, it did not precipitate significant withdrawal symptoms as compared with naloxone, even at high doses.
  • a warm-water tail-withdrawal test was used in a morphine cumulative-dosing mouse model.
  • NFP neuropeptide phosphatidylcholine
  • the pharmacological profile of NFP was further evaluated to determine its applications in opioid tolerance and opioid use disorder treatment.
  • NFP displayed opioid antagonism in the calcium flux assay. Additionally, in desensitization studies, it was shown that NFP did not produce desensitization and downregulation of the MOR. In fact, NFP showed the ability to antagonize internalization of the MOR following desensitization by opioid agonists.
  • NFP may be a P-gp substrate in vitro
  • in vivo studies demonstrated its ability to block morphine’s antinociceptive effects in a 7 day warm-water tail-withdrawal assay. Overall, these studies indicate that NFP is suitable for use as a therapeutic candidate for the treatment of opioid use disorder, without producing tolerance or inducing withdrawal symptoms.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
EP19851106.5A 2018-08-21 2019-08-19 Nicht-peptid-opioid-rezeptormodulatoren Pending EP3840751A4 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201862720552P 2018-08-21 2018-08-21
PCT/US2019/046980 WO2020041159A1 (en) 2018-08-21 2019-08-19 Non-peptide opioid receptor modulators

Publications (2)

Publication Number Publication Date
EP3840751A1 true EP3840751A1 (de) 2021-06-30
EP3840751A4 EP3840751A4 (de) 2022-04-27

Family

ID=69591082

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19851106.5A Pending EP3840751A4 (de) 2018-08-21 2019-08-19 Nicht-peptid-opioid-rezeptormodulatoren

Country Status (3)

Country Link
US (1) US11014933B2 (de)
EP (1) EP3840751A4 (de)
WO (1) WO2020041159A1 (de)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010083384A2 (en) * 2009-01-16 2010-07-22 Virginia Commonwealth University Non-peptidyl, potent, and selective mu opioid receptor antagonists
US8980908B2 (en) * 2009-01-16 2015-03-17 Virginia Commonwealth University Non-peptidyl, potent, and selective mu opioid receptor antagonists and their use in treating opioid addiction and opioid induced constipation

Also Published As

Publication number Publication date
WO2020041159A1 (en) 2020-02-27
US20200216458A1 (en) 2020-07-09
EP3840751A4 (de) 2022-04-27
US11014933B2 (en) 2021-05-25

Similar Documents

Publication Publication Date Title
JP6210993B2 (ja) Ｎｍｄａ受容体調節剤およびその使用
AU2011215704B2 (en) Secondary structure stabilized NMDA receptor modulators and uses thereof
JP6630374B2 (ja) τリン酸化を阻害する方法
JP6624594B2 (ja) 炎症性、代謝性、腫瘍性および自己免疫疾患の処置に有用なピリミジンの任意選択により縮合されているヘテロシクリル置換誘導体
JP2003505435A (ja) メラノコルチン−４受容体ゴニストとしての置換ピペリジン
HRP20110946A2 (hr) Enantiomeri spirooksindolskih spojeva i njihova upotreba kao terapijska sredstva
Mosberg et al. Opioid peptidomimetics: leads for the design of bioavailable mixed efficacy μ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist ligands
WO2015168286A1 (en) Substituted 4-phenylpiperidines, their preparaiton and use
WO2013106528A1 (en) Bivalent ligands for the treatment of neurological disorders
WO2024168098A2 (en) 5-methoxy-n,n-dimethyltryptamine analogs, their synthesis, and methods for treatment of neurological, psychiatric, and substance use disorders
EP3458061B1 (de) Potente und selektive mu-opioid-rezeptor-modulatoren
KR20140036305A (ko) 유기 화합물
AU2023204433A1 (en) Deuterated organic compounds and uses thereof
US10597378B2 (en) Tetrahydroisoquinolines for use as MOR/NOP dual agonists
US11014933B2 (en) Non-peptide opioid receptor modulators
JP6603668B2 (ja) Ｎｍｄａ受容体モジュレーター及びプロドラッグ、塩、並びにこれらの使用
US20250195504A1 (en) Naltrexamine derivatives bearing 5-member heterocyclic ring systems and opioid receptor modulators
CN120344242A (zh) 生长抑素受体亚型4(sstr4)激动剂和其应用
JP2014506565A (ja) ダイニンのキナゾリノン阻害剤
Wang et al. Synthesis and biological evaluation of a peptide-remifentanil conjugate as a novel bifunctional mu/delta-opioid receptor agonist for the treatment of pain
WO2025038406A1 (en) Design, synthesis, and biological evaluation of nitrogen-walk derivatives of nan as mu opioid receptor selective modulators
Appiani ALPHA4BETA2 NEURONAL NICOTINIC ACETYLCHOLINE RECEPTOR LIGANDS: FROM SUBTYPE SELECTIVE TO STOICHIOMETRIC ISOPHORM SELECTIVE PARTIAL AGONISM.
Akins Design and Synthesis of Salvinorin-Based Compounds for the Opioid Receptors

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20210216

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
RIC1 Information provided on ipc code assigned before grant

Ipc: C07D 489/06 20060101ALI20220113BHEP

Ipc: A61K 31/485 20060101ALI20220113BHEP

Ipc: A61K 31/44 20060101AFI20220113BHEP

A4 Supplementary search report drawn up and despatched

Effective date: 20220328

RIC1 Information provided on ipc code assigned before grant

Ipc: C07D 489/06 20060101ALI20220321BHEP

Ipc: A61K 31/485 20060101ALI20220321BHEP

Ipc: A61K 31/44 20060101AFI20220321BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20240319